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Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases

a technology of tyrosyltrna synthetase and protein fragment, which is applied in the field of discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyltrna synthetases, can solve the problems of limited diagnostic utility, expression, isolation, purification of such fragments, etc., and achieve the effect of suppressing a non-canonical activity and reducing or ameliorating symptoms

Active Publication Date: 2015-05-12
PANGU BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0570]To detect AARS polypeptides that specifically block TLR activation this assay can be modified to identify potential TLR antagonists. In this case AARS polypeptides are added to the cells at a final concentration of about 250 nM per well, (or as otherwise specified in the Examples below) 1 hour prior to adding 50 ng / mL LPS. Cells are incubated and SEAP detected as described above. PBS control wells with no LPS or AARS polypeptide alone added are used to find the basal level of TLR stimulation at the time of the measurement. Control wells are pretreated with PBS and known TLR agonists and antagonists. The ratio of the background subtracted [PBS plus LPS signal] to [AARS polypeptide plus LPS signal] is used to determine percent antagonism.

Problems solved by technology

Although a few AARS fragments have been previously shown to have non-synthetase activities, the expression, isolation, purification, and characterization of such fragments for biotherapeutic, discovery, or diagnostic utility is limited, and persons skilled in the art would not have readily appreciated such activities to associate with each member of the entire family of AARSs, or with alternative fragments.

Method used

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  • Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases
  • Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-tRNA synthetases

Examples

Experimental program
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Effect test

example 1

Identification of Proteolytic Fragments and Products of Alternative Splicing from AARSs Using Protein Topography and Migration Analysis Platform

[0627]To identify AARS fragments from cell lines, conditioned media and tissues, samples are prepared in the following ways:

[0628]Mouse macrophage (RAW 264.7), cytosol and conditioned media: Cells are treated with serum free DMEM media at a density of 15×106 cells / flasks. After 48 hours conditioned media and cell pellets are collected and processed. 200 μg of protein from secreted and cytosolic proteomic fractions are separated by SDS-PAGE and gel slices are prepared for analysis by mass spectrometry.

[0629]Mouse pancreas tissue: The pancreas from three mice are chopped, dounce homogenized, and sonicated in PBS with protease inhibitors. Cytosolic proteome is isolated by centrifugation and 200 μg of protein is separated by SDS-PAGE and gel slices are prepared for analysis by mass spectrometry.

[0630]Mouse liver tissue: Three mouse livers are ch...

example 2

Identification of Splice Variants Using Deep Sequencing

[0634]Splice variants of the aminoacyl tRNA synthetase are identified using high throughput sequencing of cDNA libraries enriched for aminoacyl tRNA synthetase transcripts. The cDNA templates are prepared from total RNA extracts of tissues such as human adult and fetal brains and enriched for aminoacyl tRNA synthetase transcripts by using primer sequences specific for all annotated exons of all annotated human aminoacyl tRNA synthetases and their associated proteins.

[0635]Human Total RNAs are obtained from Clontech. For cell line and mouse tissue samples, total RNAs are extracted using RNA Extract II Kit (MN). Genomic DNA is digested in the total RNA samples by DNAase I. To obtain mature messenger RNAs (mRNAs), the RNA samples are enriched twice by binding polyA+ RNA and digestion of RNA without 5′-cap by 5′-phosphate dependent exonuclease. Complementary DNA (cDNA) is synthesized from mature RNAs using primers that anneal to exo...

example 3

Identification of AARS Polypeptides Using Bioinformatics

[0638]AARS protein fragments (resectin or appendacrine peptides) are identified using bioinformatics. Amino acid sequences of the full length human aminoacyl tRNA synthetase are aligned with the full length amino acid sequence of its ortholog from the bacterium Escherichia coli using a program such as FASTA (available at the website http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi) or the BLASTP program from the NCBI (available at the website http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi?PROGRAM=blastp&BLAST PROGRAM S=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK LOC=blasthom). Resectin sequences from the human proteins are identified as sequences covering regions where there are gaps in the bacterial sequence in the alignment, or regions with low homology between the two species. The peptide, and corresponding DNA sequences in Table 3 include examples identified in this way.

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Abstract

Provided are compositions comprising newly identified protein fragments of aminoacyl-tRNA synthetases, polynucleotides that encode them and complements thereof, related agents, and methods of use thereof in diagnostic, drug discovery, research, and therapeutic applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a U.S. national phase application of International Patent Application No. PCT / US2011 / 049223 filed on Aug. 25, 2011, which claims the benefit under 35 U.S.C. §119(e) of U.S. provisional patent application No. 61 / 377,006 filed on Aug. 25, 2010, and U.S. provisional patent application No. 61 / 488,616 filed on May 20, 2011, the entire contents of each of which, are incorporated herein by reference.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 120161—489PC_SEQUENCE_LISTING.txt. The text file is about 62 KB, was created on Aug. 18, 2011, and is being submitted electronically via EFS-Web.TECHNICAL FIELD[0003]The present invention relates generally to compositions comprising newly identified protein fragm...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K38/00C07K1/00A61K38/16C12N9/00
CPCA61K38/16C12Y601/01001C12N9/93A61K38/00C12Y601/01A61P21/00A61P25/00A61P29/00A61P3/00A61P31/00A61P35/00A61P37/02A61P37/06A61P9/00A61P3/10C07K16/40G01N33/573C07K2317/92G01N2333/9015C07K2317/24C07K2317/622
Inventor GREENE, LESLIE ANNCHIANG, KYLE P.HONG, FEIVASSEROT, ALAIN PHILIPPELO, WING-SZEWATKINS, JEFFRY D.QUINN, CHERYL L.MENDLEIN, JOHN D.
Owner PANGU BIOPHARMA
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